GASTRULA PERIOD (5 1/4 - 10 h)

Epiboly continues, and in addition, the morphogenetic cell movements of involution, convergence, and extension occur, producing the primary germ layers and the embryonic axis.

The beginning of involution defines the onset of gastrulation, and, so far as we have been able to tell, this occurs at 50%-epiboly (Fig. 11A). As a consequence, within minutes of reaching 50%-epiboly a thickened marginal region termed the germ ring appears, nearly simultaneously all around the blastoderm rim (Fig. 11B, arrow in C). Convergence movements then, nearly as rapidly, produce a local accumulation of cells at one position along the germ ring, the so-called embryonic shield (Fig. 11D, arrow in E). During these events, epiboly temporarily arrests, but after the shield forms, epiboly continues; the margin of the blastoderm advances around the yolk cell to cover it completely (Fig. 11F-L). The advance occurs at a nearly constant rate, over an additional 15% of the yolk cell each hour, and providing a useful staging index during most of gastrulation (Fig. 12).

Just as there was no blastocoele during the blastula period, there is no archenteron in the gastrula. Neither is there a blastopore; DEL cells involute at the blastoderm margin, which thus plays the role of a blastopore. Involution produces the germ ring by folding the blastoderm back upon itself. Hence, within the germ ring there are two germ layers, the upper, the epiblast, continues to feed cells into the lower, the hypoblast, throughout gastrulation. Note that these terms, epiblast and hypoblast, are also used to describe layers of the avian embryonic blastoderm, but the layers so-named seem to be altogether different in these two kinds of vertebrate embryos.

As a morphogenetic movement, involution at the blastoderm margin seems particularly well documented for the rosy barb (Wood and Timmermans, 1988), a small teleost that develops in many ways very similarly to the zebrafish. However, recently J. P. Trinkaus has obtained evidence (unpublished) that in Fundulus, a somewhat larger embryo, involution may not occur at all; rather, cells near but usually not just at the margin move from shallow to deeper positions as individuals, rather than in a rolling cell layer. This movement, termed ingression, could also happen in zebrafish (J. Shih and S. E. Fraser, unpublished observations), an issue for further study by high-resolution time-lapse analysis of whole cell fields of the sort done by Wood and Timmermans.

As a consequence of the inward movement, a fissure, Brachet's cleft (Ballard, 1980), becomes visible with Nomarski optics (Fig. 13), or in sectioned material, between the epiblast and hypoblast. We point out the location of the cleft in the drawing of the 75%-epiboly stage in Fig. 1, although one cannot always locate it easily with the dissecting microscope (compare Fig. 11 and Fig. 13). Cells may not normally mix between the epiblast and hypoblast across Brachet's cleft (although they are able to so mix an experimental situation; see below). Cells in the two layers are streaming in different directions. Except for the dorsal region (see below), the epiblast cells, generally stream towards the margin, and those reaching the margin move inwards to enter the hypoblast. Then, as hypoblast cells, they stream away from the margin (Warga and Kimmel, 1990). The cells remaining in the epiblast when gastrulation ends correspond to the definitive ectoderm and will give rise to such tissues as epidermis, the central nervous system, neural crest, and sensory placodes. The hypoblast gives rise to derivatives classically ascribed to both the mesoderm and endoderm; Thisse et al. (1993) call the same layer "mesendoderm". Both names emphasize the absense of three separate germ layers at any stage during the gastrula period, only two are present. The hypoblast is only about one or two cells thick around most of the circumference of the embryo (Fig. 13), and it is presently unknown how this layer subdivides into endoderm and mesoderm.

In the manner of lineage restrictions to the EVL that occurred at sphere stage at 4h, about an hour later many but not all DEL cells become lineage-restricted to one or another tissue or organ-specific fate. The restriction occurs about the time gastrulation begins (Kimmel and Warga, 1986; Kimmel et al., 1990b, 1994), and as for the earlier EVL restriction, this restriction of DEL cells in the early gastrula seems unaccompanied by cell commitment (Ho and Kimmel, 1993): If DEL cells are transplanted, as single cells, from the marginal region that normally generates mesodermal or endodermal fates, to the animal pole region that normally forms ectodermal fates, they plastically regulate both their morphogenetic movements and their eventual fates; appearing indistinguishable from their new neighbors in both respects. The ability to developmentally regulate may involve dynamic regulation of the activities of zygotically expressed patterning genes: Marginal cells specifically express the gene no tail (ntl), the homologue of mouse T/Brachyury, that codes a nuclear protein (Schulte-Merker et al., 1992) essential for normal notochord development (Halpern et al., 1993; Schulte-Merker et al., 1994). Cells transplanted from the margin to the animal pole rapidly turn off ntl transcription, and cells transplanted in the opposite direction just as rapidly turn it on (Schulte-Merker et al., 1992). However, by the middle of gastrulation the ability of the cells to regulate so plastically seems to be lost, because single cells transplanted from the hypoblast to epiblast at this stage will rapidly leave their new positions, re-enter the hypoblast, and develop a hypoblast-derived fate appropriate for just where they end up. To exhibit this behavior, the transplanted cells have to cross Brachet's cleft, and time-lapse analysis reveals that they accomplish this unusual relocation apparently without impediment (Ho and Kimmel, 1993).

An organ- and tissue-level fate map is available for the onset of gastrulation (Fig. 14). The map is made by injecting single cells with lineage-tracer dye, and later ascertaining in what region of what organ the labeled descendants of the injected cell lie, and generally what differentiated type or types they have formed, as determined from the cell shapes, sizes, and positions. Topologically the fate map is broadly equivalent to fate maps of other chordates, notably amphibians (Keller, 1975; 1976; Dale and Slack, 1987), and also birds (Hatada and Stern, 1994) and mammals (Lawson et al., 1991). Within the DEL, primary germ layers are mapped according to latitude (position relative to the animal pole and the margin), and tissue rudiments can be located with reference to both cell latitude and longitude (the position relative to the dorsal midline). Another notable feature, not apparent in the figure, is that position along the dorsal-ventral (DV) axis of the gastrula generally corresponds to later position along the anterior-posterior (AP) axis of the pharyngula. This is the case for all three germ layers; eventual AP cell positions in the spinal cord (ectoderm), muscle (mesoderm) and gut wall (endoderm) all correlate with DV cell position in the early gastrula. Morphogenesis to make this DV-AP transform is complicated and largely not understood. Consider the neurectodermal fate map: We see complexity in that not only do the more anterior neural fates map to the dorsal side of the gastrula, but also the more theventral cell types of the neural tube, the motoneurons and floor plate, map to the same region -- the dorsal side of the gastrula (Kimmel et al., 1990b). We discuss later how dorsal cells in the gastrula eventually become ventral cells in the neural tube, but just how dorsal versus anterior fates sort out in this region of the fate map is simply unknown. Scattering of clonally related cells (and hence positionally related cells) during morphogenesis during and after gastrulation generally precludes very fine-grained mapping, but this is not always the case. For example, Stanier et al. (1993) have shown that single late blastula cells contribute projeny to either the heart's atrium or ventricle but not do not scatter such that a single clone encompasses both chambers.

EVL cells neither divide (Kimmel et al., 1994) nor involute, nor do they appear to converge, at least very much, during gastrulation. They continue to flatten, and they relocate by epiboly towards the vegetal pole. Irrespective of their early positions, the EVL cells differentiate directly as periderm, an extremely flattened protective outer single-cell layer covering the entire embryo. The position of an EVL cell in the early gastrula fairly reliably predicts just where in the periderm of a later embryo a descendent clone will be located, which is useful for certain cell lineage and fate map analyses (Kimmel et al., 1990b).

Near the time that involution begins, other DEL cell movements and rearrangements occur both within the epiblast and hypoblast. During convergence, cells stream from all sectors of the blastoderm towards the dorsal side. Intercalations repack the cells in both layers. In the late blastula the intercalations were radial, and accompanied blastoderm thinning (epiboly). The new intercalations are mediolateral (Keller and Tibbetts, 1989); they effect convergent extension, a narrowing and elongation of the primary embryonic axis. As gastrulation proceeds the cells undergo many such directional intercalations, such that single labeled clones descended from marked blastula founders become markedly spread apart. They form into long discontinuous strings of cells, oriented longitudinally (Warga and Kimmel, 1990b; Kimmel et al., 1994).

Formation of the embryonic shield visibly indicates that rapid convergence movements have begun. The shield is an accumulation and condensation of DEL cells along a local stretch of about 30 degrees of longitude of the germ ring, and marks the future dorsal side of the embryo. Hence, when the shield forms one can reliably distinguish the orientation of the embryo's eventual DV axis. Cells involuting at the shield form the so-called "axial" hypoblast. Expression of the gene goosecoid is a reliable marker of where the shield will form, and appears to label the earliest cells to involute in the axial hypoblast (Stachel et al., 1993). These first cells, expressing goosecoid, stream anteriorwards, and appear to correspond to precursors of the tetrapod prechordal plate. A principal derivative of the prechordal plate is the hatching gland, and the endodermal lining of the pharynx comes from the same region. Figure 14 does not show these fates; they lie at the margin just beneath the notochord region. Cells of the notochord rudiment, the chorda mesoderm, then begin to involute at the dorsal midline only shortly after these earliest prospective prechordal plate cells, as the embryonic shield becomes more easily visible.

Convergence and extension, involving cell mixing, and anteriorwards cell migration is extreme within the embryonic shield and surrounding region. DEL cells in both layers of the early shield distribute along the entire length of the AP axis, from the pericardial region, ventral to the head, to the end of the tail. For example, axial cells in the shield epiblast can relocate anteriorwards to form ventral forebrain (K. Hatta, unpublished observations), becoming new neighbors of cells originally located at the animal pole, for much of the forebrain fate maps to the animal pole of the early gastrula (Kimmel et al., 1990b). Anteriorwards movement in the axial hypoblast of cells deriving from the shield is similarly remarkable: To form the hatching gland, located on the pericardium (on the yolk sac ventral to the head), prechordal plate cells must first move anteriorwards along the dorsal midline. Then they move under and in front of the forebrain anlagen, where, as gastrulation ends, they form a prominent pile known as the polster (arrow at the bud stage in Fig. 1 and upper arrow in Fig. 11L). Eventually the hatching gland forms on the pericardial membrane, ventral to the head (arrow at the prim-6 stage in Fig. 1). However, this last change, from a rostral to a ventral location, probably comes about with no real ventralwards movement of these cells at all. Rather, head straightening that begins during the segmentation period (discussed in detail below), would serve to lift the head rudiment upwards dorsally and away from the position of the hatching gland primordium, where these axial mesodermal cells might passively remain.

Axial mesoderm appears to have the potential to induce a second embryonic axis, as revealed in transplantation experiments involving cells of the embryonic shield of the early gastrula (Ho, 1992b). Hence, the embryonic shield, in terms of its inductive role demonstrated in these experiments, its dorsal marginal position in the embryo, and the fates it eventually generates, is broadly equivalent to the amphibian organizer region of Spemann.

As epiboly resumes (roughly an hour and a half after the beginning of gastrulation) and the shield extends towards the animal pole, one can clearly see that the blastoderm is prominently thicker on the dorsal side than on the ventral side (Fig. 11F-H, J). This seems to be due to the presence dorsally of axial hypoblast, for sections through the gastrula reveal that the epiblast does not appear regionally distinctive until late in the gastrula period (Schmitz et al., 1993; R. M. Warga, unpublished observations). About midway through gastrulation, the axial hypoblast becomes clearly distinct from paraxial hypoblast, that flanks it on either side (Fig. 11I). Anterior paraxial hypoblast will generate muscles to move the eyes, jaws, and gills. More posteriorly, much of the paraxial hypoblast is present as the segmentalplate that will form somites.

The dorsal epiblast begins to thicken rather abruptly near the end of gastrulation, the first morphological sign of development of the rudiment of the central nervous system rudiment, the neural plate. The thickening occurs anteriorly, and initially just at the midline, where the epiblast overlies axial mesoderm. The lateral borders of the neural plate remain morphologically indistinct at this time, in sectioned material (Schmitz et al., 1993) as well as in the living embryo.

We define the gastrula period as ending when epiboly is complete, and the tail bud has formed Fig. 11. However, this is purely operational, because gastrulation movements seem to continue in the tail bud after this time. DEL cells typically pass through cell cycle 15 and enter cycle 16 during gastrulation. The YSL is postmitotic, and the EVL cells typically are in a long interphase of cycle 14.